Culturing Fastidious Prokaryotic Vascular Plant Pathogens · 2020. 10. 20. · babaco or mountain...
Transcript of Culturing Fastidious Prokaryotic Vascular Plant Pathogens · 2020. 10. 20. · babaco or mountain...
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Culturing Liberibacter
Michael J. Davis
Citrus Research and Education Center
UF/IFAS
Lake Alfred, Florida
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Some Direct and Indirect Advantages of Culturing Liberibacter spp.
• Facilitate development methodologies for detection and identification of the bacteria and greening disease
• Facilitate studies on greening disease epidemiology
• Facilitate studies on host/pathogen/insect vector interactions.
• Facilitate studies on genomics and molecular biology of Liberibacter.
• Facilitate development of procedures to manage greening disease.
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Fastidious Prokaryotic Plant Pathogens
Xylem-Limited
Xylella fastidiosa
Clavibacter xyli subsp. xyli
Clavibacter xyli subsp. cynodontis
Papaya Bunchy Top Rickettsia
Laticifer-Inhabiting
Phloem-Limited
Spiroplasma citri
Spiroplasma kunkelii
Candidatus Phytoplasma spp.
Candidatus Liberibacter spp.
Spiroplasma phoeniceum
Unnamed Bacteria
Candidatus Phlomobacter fragariae
Candidatus Phlomobacter betae
Serratia marcescens
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The Genus Spiroplasma JOSEPH G. TULLY and ROBERT F. WHITCOMB
The Prokaryotes
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Media for Spiroplasmas
BSR Medium (Bové et al., 1978), per literBeef heart infusion 25 gGlucose 1 gFructose 1 gSucrose 10 gSorbitol 70 gPhenol red 20 mgDeionized water 900 mlHorse serum 100 ml
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Fastidious Bacteria of Plant Vascular Tissues and Their Invertebrate Vectors M. J. DAVIS
The Prokaryotes
Clavibacter xyli subsp. xyliR. H. LAWSON
Fastidious
Xylem-limited
Bacteria
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PW Medium for Xylella fastidiosa______________________________________________________________Pancreatic digest of casein 1 gPapaic digest of soy meal 4 gHemin chloride 10 mgMgSO4·7H2O 0.4 gK2HPO4 1 gKH2PO4 1.2 gAgar 12 gPhenol red 20 mgDistilled Water 1 literBovine serum albumin fraction five 6 gGlutamine 4 g______________________________________________________________Davis M. J. ; B. C. Raju; R. H. Brlansky; R. F. Lee; L. W. Timmer; R. C.
Norris; R. E. McCoy; 1983. Periwinkle wilt bacterium: Axenic culture, pathogenicity, and relationships to other Gram-negative, xylem-inhabiting bacteria. Phytopathology, vol. 73, pp. 1510–1515
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SC Medium for Isolating Clavibacter xyli
________________________________________________________Papaic digest of soy meal 8 gHemin chloride 10 mgMgSO4·7H2O 0.2 gK2HPO4 0.5 gKH2PO4 1.5 gCorn meal agar 17 gDistilled Water 1 literGlucose 0.5 gCysteine 1 gBovine serum albumin fraction five 2 g________________________________________________________Davis M. J. ; A. G. Gillaspie, Jr.; R. W. Harris; R. H. Lawson;
1980a. Ratoon stunting disease of sugarcane: Isolation of the causal bacterium. Science, vol. 210, pp. 1365–1367
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California Buckeye Leaf Scorch
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BLAST Search Results for Buckeye Bacterium 16S rRNA Gene Sequence
Score E Sequences producing significant alignments: (Bits) Value gi|20378103|gb|AF373185.1| Pectobacterium carotovorum subsp. ... 2557 0.0 gi|20378107|gb|AF373189.1| Pectobacterium carotovorum subsp. ... 2553 0.0 gi|20378105|gb|AF373187.1| Pectobacterium carotovorum subsp. ... 2549 0.0 gi|20378121|gb|AF373203.1| Pectobacterium chrysanthemi strain... 2533 0.0 gi|20378101|gb|AF373183.1| Pectobacterium carotovorum subsp. ... 2524 0.0 gi|20378106|gb|AF373188.1| Pectobacterium carotovorum subsp. ... 2520 0.0 gi|38373979|gb|AY363386.2| Klebsiella sp. rennanqilfy18 16S r... 2494 0.0 gi|71480490|gb|DQ068854.1| Uncultured bacterium clone 6s27 16... 2486 0.0 gi|4582000|emb|AJ233411.1|ECA233411 Erwinia carotovora 16S rRNA 2482 0.0 gi|20378104|gb|AF373186.1| Pectobacterium carotovorum subsp. ... 2478 0.0 gi|2584742|emb|Z96089.1|ECZ96089 Erwinia carotovora LMG 2404-T 1 2476 0.0 gi|56159736|gb|AY825036.1| Enterobacter aerogenes strain NTG-... 2470 0.0 gi|71480420|gb|DQ068784.1| Uncultured bacterium clone f1s5 16... 2464 0.0 gi|51243827|gb|AY690721.1| Erwinia sp. MK14 16S ribosomal RNA ge 2462 0.0 gi|71480418|gb|DQ068782.1| Uncultured bacterium clone f1s3 16... 2462 0.0 gi|6562390|emb|AJ251468.1|EAE251468 Enterobacter aerogenes parti 2462 0.0
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Papaya Bunchy Top Disease
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BLAST Search Results for the Papaya Bunchy Top Rickettsia 16S rRNA Gene Sequence
Score E Sequences producing significant alignments: (Bits) Value gi|506778|gb|U11014.1|RBU11014 Rickettsia bellii 16S rRNA gene, 2789 0.0 gi|535750|gb|L36103.1|RIRRGDF Rickettsia bellii 16S ribosomal RN 2763 0.0gi|1147763|gb|U42084.1|RPU42084 Rickettsia PAR 16S ribosomal RNA 2759 0.0gi|58978283|gb|AY753175.1| Rickettsia endosymbiont of Tetrany... 2728 0.0gi|67003925|gb|CP000053.1| Rickettsia felis URRWXCal2, complete 2718 0.0gi|15620042|gb|AE008647.1| Rickettsia conorii str. Malish 7, ... 2686 0.0 gi|506787|gb|U11021.1|RRU11021 Rickettsia rickettsii 16S rRNA ge 2686 0.0gi|535749|gb|L36102.1|RIRRGDE Rickettsia sp. 16S ribosomal RNA ( 2680 0.0gi|70568364|dbj|AB196668.1| Rickettsia sp. PAR-OY gene for 16... 2678 0.0 gi|600169|gb|U17645.1|U17645 Rickettsia honei 16S ribosomal RNA 2678 0.0 gi|535753|gb|L36106.1|RIRRGDI Rickettsia massiliae 16S ribosomal 2676 0.0 gi|538433|gb|L36214.1|RIRRGDM Rickettsia massiliae (strain Mt... 2676 0.0 gi|33438751|gb|AF178037.2| Rickettsia heilongjiangii 16S ribo... 2674 0.0 gi|6979193|gb|AF120026.1| Rickettsia sp. RpA4 strain RpA4 16S... 2672 0.0 gi|31323700|gb|AF510102.1| Rickettsia sp. Chad 16S ribosomal RNA 2670 0.0 gi|600168|gb|U17644.1|RAU17644 Rickettsia australis 16S ribosoma 2670 0.0 gi|538435|gb|L36216.1|RIRRGDO Rickettsia rhipicephali (strain... 2668 0.0 gi|529688|gb|U12460.1|RCU12460 Rickettsia conorii ITT-597 16S rR 2666 0.0
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Babaco Bacterium• Unidentified bacterium isolated from
babaco or mountain papaya (hybrid of Carica stipulata and C. pubescens).
• Bacterium is very fastidious, and grows on media developed for the buckeye bacterium but not those developed for other fastidious bacteria.
• Is an alpha proteobacterium based on its 16S rRNA gene sequence.
• Not the papaya bunchy top rickettsia– 16S rRNA sequences differ– PBT bacterium did not grow on media
developed for the Babaco bacterium
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BLAST Search Results for Babaco Bacterium 16S rRNA Gene Sequence
Score E Sequences producing significant alignments: (Bits) Value gi|57918592|gb|AY742824.1| Candidatus Liberibacter americanus... 1961 0.0gi|60267995|gb|AY919312.1| Candidatus Liberibacter sp. LSg2 1... 1503 0.0 gi|38426928|gb|AY444978.1| Uncultured bacterium clone M16 16S... 1427 0.0 gi|2290246|gb|U87772.1|AGU87772 Afipia genosp. 6 16S ribosomal R 1417 0.0 gi|15430883|gb|AF407383.1| Uncultured bacterium clone RB7C4 1... 1409 0.0 gi|15072624|emb|AJ278726.1|EST278726 Methylocapsa acidiphila ... 1368 0.0 gi|73698030|gb|DQ157274.1| Candidatus Liberibacter asiaticus ... 1348 0.0 gi|73698029|gb|DQ157273.1| Candidatus Liberibacter asiaticus ... 1348 0.0 gi|2570146|dbj|AB008366.1| Liberibacter sp. gene for 16S rRNA, p 1348 0.0 gi|73698031|gb|DQ157275.1| Candidatus Liberibacter asiaticus ... 1340 0.0 gi|28397343|gb|AY192576.1| Candidatus Liberibacter asiaticus ... 1332 0.0 gi|438690|gb|L22533.1|LIE16SRRNB Liberibacter africanus 16S ribo 1330 0.0 gi|60267994|gb|AY919311.1| Candidatus Liberibacter asiaticus ... 1324 0.0 gi|11125697|dbj|AB038369.1| Liberibacter endosymbiont of Diap... 1312 0.0 gi|438689|gb|L22532.1|LIE16SRRNA Liberibacter asiaticus 16S ribo 1300 0.0 gi|46361651|gb|AY500255.1| Sinorhizobium sp. ORS 1400 16S rib... 1259 0.0 gi|58294523|gb|AY875975.1| Sinorhizobium sp. ORS3178 16S ribo... 1251 0.0 gi|21952355|gb|AF523952.1| Uncultured bacterium clone FW8 16S... 1223 0.0
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Significance of the Babaco Bacterium
• The Babaco bacterium appears to be the most closely related species to the Liberibacter spp. that can be grown in axenic culture at the present time.
• It follows that the Babaco bacterium might be used in a “model system” for the development of media and procedures for culturing Liberibacter spp.
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Isolation in Culture of Liberibacter
• Numerous media were developed that support growth of the Babaco bacterium and the corn stunt spiroplasma
• Media were tested for supporting growth of Liberibacter
• Fastidious bacteria isolated from two of 64 extracts from HLB infected citrus
• Cultures were PCR-positive for Liberibacter
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Liberibacter Cultures• Mixed culture: The Liberibacter is growing
in association with other bacteria.• Single colony isolations retain mixed
culture at a high frequency.– Suggests that both bacteria benefit from the
presence of the other.• Second bacterium overshadows
Liberibacter.– PCR detection of Liberibacter is often weak
and inconsistent
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Present Efforts• Attempting to modify medium and culture
conditions to support better growth of Liberibacter
• Will attempt to use second bacterium to establish new cultures of Liberibacter
• Will determine if cell-free supernatants from cultures of the second bacterium support growth of Liberibacter